JP7455316B2 - Anticancer drugs, prodrugs of anticancer drugs, methods for killing cancer cells outside the body, cancer treatment methods, and cancer treatment devices - Google Patents

Description

The present invention relates to an anticancer drug, a prodrug of an anticancer drug, a method for killing cancer cells outside the body, a method for treating cancer, and a device for treating cancer.

Approximately 20,000 people are treated annually at proton beam therapy facilities around the world, with a total of over 210,000 patients. However, there are 17 million cancer patients in the world, and only 1 in 850 patients receive cancer treatment using proton beams.

Patent No. 4520219 Patent No. 7648586 Patent No. 5392752

F. Tabbakh, N. S. Hosmane, Enhancement of Radiation Effectiveness in Proton Therapy: Comparison Between Fusion and Fission Methods and Further Approaches, Scientific Reports 10 (2020) 5466. Takashi Tanaka et al., Production of by-products in 5-aminolevulinic acid production using photosynthetic bacterial mutants and mass production of 5-aminolevulinic acid by controlling culture temperature, Journal of the Japan Society of Biotechnology 93 (2015) 24-31. C. ROLFS and W. S. RODNEY, PROTON CAPTURE BY 15N AT STELLAR ENERGIES, Nuclear Physics, A235 (1974) 450-459. G. Imbriani, et al., Measurement of γ rays from 15N(p,γ)16O cascade and 15N(p,α1γ)12C reactions, PHYSICAL REVIEW, C85 (2012) 065810. Technical data “Torelina (registered trademark) Torelina (registered trademark) polyphenylene sulfide film” Toray Industries, Inc. Industrial Materials Division 1 (2013). M. Tamura, H. Ito, H. Matsui, Radiotherapy for cancer using X-ray fluorescence emitted from iodine, Scientific Reports 7 (2017) 43667. M Shibuta, et al., Imaging cell picker: A morphology-based automated cell separation system on a photodegradable hydrogel culture platform, J. Bioscience and Bioengineering, 126 (2018) 653-660.

According to the findings of the present inventors, if the survival rate of cancer patients by proton beam therapy increases, proton beam therapy can be expected to become widespread. According to the findings of the present inventors, proton beam therapy can be used to treat pancreatic cancer in Japanese men at sites that are conventionally difficult to cure, with an extremely low 5-year survival rate (8.5%, 2009-2011). Other causes of death are by far the highest (24.2% of 220,339 male cancer deaths in 2019) Lung cancer, grade 4 brain tumor symptoms, and glioblastoma with a low 5-year survival rate of less than 10% It can be expected to increase the survival rate not only for cancers specific to organs such as cancer patients, but also for patients with cancers such as lymphoma and dissemination, which are feared by causing cancer metastasis. Furthermore, according to the findings of the present inventors, proton beam therapy can be expected to be applied to breast cancer, for which non-invasive treatment is highly desired.

According to the findings of the present inventors, it is desirable to specifically kill cancer cells in order to increase the survival rate of cancer patients and to treat cancer efficiently in a non-invasive manner. Therefore, an object of the present invention is to provide an anticancer drug that specifically kills cancer cells, a prodrug of the anticancer drug, a method for killing cancer cells in vitro, a method for treating cancer, and a device for treating cancer. shall be at least a part of.

[1] An anticancer agent containing a substance that specifically causes nitrogen-15 to accumulate in cancer cells.

[2] The anticancer agent according to [1], which is administered into a patient's body, and after administration, the patient is irradiated with a proton beam.

[3] Substance that specifically accumulates nitrogen-15 in cancer cells for use in cancer treatment

[4] The substance according to [3], which is administered into a patient's body, and after administration, the patient is irradiated with a proton beam.

[5] The anticancer agent according to [1], which is a molecular target therapeutic agent in which the nitrogen is nitrogen-15.

[6] The anticancer agent according to [5], which contains a portion that binds to cancer cells.

[7] The anti-cancer agent according to [6], wherein the portion that binds to cancer cells is an antibody or a part of an antibody.

[8] The anticancer agent according to [7], wherein a drug is bound to the antibody or a part of the antibody.

[9] The anticancer agent according to [7], wherein the antibody or a part of the antibody is trastuzumab in which nitrogen is nitrogen-15.

[10] Use of a substance that causes nitrogen-15 to specifically accumulate in cancer cells for the production of an anticancer drug.

[11] The use according to [10], wherein the anticancer drug is administered into a patient's body, and after administration, the patient is irradiated with a proton beam.

[12] An anticancer agent containing 5-aminolevulinic acid whose nitrogen is nitrogen-15.

[13] The anticancer agent according to [12], which is administered into a patient's body, and after administration, the patient is irradiated with a proton beam.

[14] 5-Aminolevulinic acid whose nitrogen is nitrogen-15 for use in the treatment of cancer.

[15] The 5-aminolevulinic acid according to [14], wherein the nitrogen is nitrogen-15, which is administered into a patient's body and the patient is irradiated with a proton beam after administration.

[16] Use of 5-aminolevulinic acid whose nitrogen is nitrogen 15 for the production of an anticancer drug.

[17] The use according to [16], wherein the anticancer drug is administered into a patient's body, and after administration, the patient is irradiated with a proton beam.

[18] An anticancer agent containing 5-fluorouracil in which nitrogen is nitrogen-15.

[19] The anticancer agent according to [18], which is administered into a patient's body, and after administration, the patient is irradiated with a proton beam.

[20] 5-fluorouracil in which the nitrogen is nitrogen-15 for use in the treatment of cancer.

[21] The 5-fluorouracil according to [20], wherein the nitrogen is nitrogen-15, which is administered into a patient's body and the patient is irradiated with a proton beam after administration.

[22] Use of 5-fluorouracil in which the nitrogen is nitrogen-15 for the production of an anticancer agent.

[23] The use according to [22], wherein the anticancer drug is administered into the body of a patient, and after administration, the patient is irradiated with a proton beam.

[24] A prodrug of an anticancer drug containing a substance that causes nitrogen-15 to specifically accumulate in cancer cells.

[25] The prodrug according to [24], which is administered into a patient's body, and after administration, the patient is irradiated with a proton beam.

[26] A prodrug of an anticancer agent containing a substance that causes nitrogen-15 to specifically accumulate in cancer cells, for use in cancer treatment.

[27] The prodrug according to [26], which is administered into the body of a patient, and after administration, the patient is irradiated with a proton beam.

[28] Use of a substance that causes nitrogen-15 to be specifically accumulated in cancer cells for the production of a prodrug of an anticancer drug.

[29] The use according to [28], wherein the anticancer drug prodrug is administered into the body of a patient, and after administration, the patient is irradiated with a proton beam.

[30] A prodrug of an anticancer drug containing 5-fluorouracil in which nitrogen is nitrogen-15.

[31] The prodrug according to [30], which is administered into a patient's body, and after administration, the patient is irradiated with a proton beam.

[32] The prodrug according to [30] or [31], which is selected from the group consisting of tegafur, tegafur-uracil, tegafur-gimeracil-oteracil potassium, doxifluridine, and capecitabine, in which nitrogen is nitrogen 15.

[33] A prodrug of an anti-cancer drug containing 5-fluorouracil in which the nitrogen is nitrogen-15 for use in the treatment of cancer.

[34] The prodrug according to [33], which is administered into a patient's body, and after administration, the patient is irradiated with a proton beam.

[35] The prodrug according to [33] or [34], which is selected from the group consisting of tegafur, tegafur uracil, tegafur gimeracil oteracil potassium, doxifluridine, and capecitabine, in which nitrogen is nitrogen 15.

[36] Use of any one selected from the group consisting of tegafur, tegafur uracil, tegafur gimeracil oteracil potassium, doxifluridine, and capecitabine, in which nitrogen is nitrogen 15, for the production of a prodrug of an anticancer drug. .

[37] The use according to [36], wherein the anticancer drug prodrug is administered into the body of a patient, and after administration, the patient is irradiated with a proton beam.

[38] A method for killing cancer cells outside the body, comprising (a) accumulating nitrogen-15 in the cancer cells outside the body, and (b) irradiating the cancer cells with a proton beam outside the body.

[39] The method for killing cancer cells in vitro according to [38], wherein accumulating nitrogen-15 in cancer cells includes providing the cancer cells with any of the anticancer agents described above.

[40] Killing cancer cells in vitro according to [38], wherein accumulating nitrogen-15 in cancer cells comprises providing the cancer cells with a prodrug of the anticancer drug according to any of the above. Method.

[41] A method for treating cancer, comprising (a) accumulating nitrogen-15 in cancer cells of a human or non-human animal, and (b) irradiating the human or non-human animal with a proton beam.

[42] The method according to [41], wherein accumulating nitrogen-15 in cancer cells of a human or non-human animal comprises administering to the human or non-human animal any of the anticancer agents described above. How to treat cancer.

[43] Accumulating nitrogen-15 in cancer cells of a human or non-human animal comprises administering to the human or non-human animal a prodrug of any of the anticancer agents described above [41] The cancer treatment method described in .

[44] A cancer treatment device comprising an irradiation device for irradiating a human or non-human animal with proton beams in which nitrogen-15 has accumulated in cancer cells.

[45] The cancer treatment device according to [44], wherein the human or non-human animal is administered any of the anticancer agents described above.

[46] The cancer treatment device according to [44], wherein the human or non-human animal is administered a prodrug of any of the anticancer agents described above.

[47] A cancer treatment device described in any one of [44] to [46], further comprising an accelerator for accelerating a proton beam.

[48] The cancer treatment device according to [47], wherein the accelerator includes laser plasma.

According to the present invention, it is possible to provide an anticancer drug that specifically kills cancer cells, a prodrug of the anticancer drug, a method for killing cancer cells outside the body, a method for treating cancer, and a device for treating cancer.

FIG. 2 is a schematic diagram showing the pathway by which protoporphyrin IX derived from 5-aminolevulinic acid is converted to heme. ¹⁵ is a schematic diagram showing resonance energy positions due to a 15 N( ¹ H, α ₁ γ) ¹² C resonance nuclear reaction. FIG. ¹⁵ is a diagram showing the relationship between a laboratory system and a centroid system in a 15 N( ¹ H, α ₁ γ) ¹² C resonance nuclear reaction. FIG. 1 is a diagram comparing the LET of protons and product ions. ¹⁵ is a diagram showing the proton energy dependence of the reaction cross section of 15 N( ¹ H, α ₁ γ) ¹² C resonance nuclear reaction. FIG. Describe the relationship between the resonance energy of the ¹⁵ N ( ¹ H, α ₁ γ) ¹² C resonance nuclear reaction and the reaction position (residual range) of protons in the body, and the range of the reaction generated ions on the scale of the residual range. This is a diagram. ¹⁵ N( ¹ H, α ₁ γ) ¹² C resonance energy of resonance nuclear reaction, residual range of protons in the body, range of reaction product ions ( ⁴ He ²⁺ , ¹² C ⁶⁺ ), Biological ionization effect (Bragg's peak) is shown. FIG. 1 is a schematic diagram showing that ^{a 15} N resonance nuclear reaction occurs in cancer cells. FIG. 2 is a diagram showing the thermal hydrolysis resistance of Torelina (registered trademark) (see Non-Patent Document 8). It is a table showing the gas permeability of Torelina (registered trademark) film (see Non-Patent Document 9). FIG. 1 is a schematic diagram showing a cancer treatment device according to an embodiment. FIG. 2 is a diagram showing a method for quantifying ¹⁵ N accumulated in each of normal cells and cancer cells according to Example 1. 2 is a graph showing gamma ray spectra obtained by quantifying ¹⁵ N accumulated in normal cells and cancer cells, respectively, according to Example 1. 2 is a graph showing the uptake amount of ¹⁵ N_5-ALA in normal cells and cancer cells according to Example 1. FIG. FIG. 2 is a diagram of the gene structure of the pUC4-KIXX plasmid according to Example 2. 3 is a photograph showing colonies of E. coli when the isotopic ratio of ¹⁵ N in pUC4-KIXX according to Example 2 was changed. The number of E. coli bacteria (left vertical axis) when changing the isotope ratio of ¹⁵ N in pUC4-KIXX according to Example 2, and ^{the 15} N ( ¹ H, α ₁ γ) ¹² C resonance nuclear reaction (E _r It is a graph showing the gamma ray dose (right vertical axis) of 4.43 MeV emitted at 0.987 MeV). 3 is a photograph of RGM-GFP cells cultured in a medium not containing 15N_5-ALA immediately after proton beam irradiation according to Example 3. FIG. 2 is a graph illustrating the survival rate of RGM-GFP cells cultured in a medium not containing 15N_5-ALA according to Example 3 with respect to the charge amount of proton beam irradiation. FIG. 3 is a photograph of RGK-KO cells immediately after proton beam irradiation according to Example 3. These are photographs of cells cultured in a medium containing 15N_5-ALA and a medium not containing 15N_5-ALA. These are a photograph of RGK-KO cells 24 hours after proton beam irradiation according to Example 3, and a photograph of cells cultured in a medium containing 15N_5-ALA. 3 is a photograph of a cell holder that allows cells to be kept alive in vacuum according to Example 4. 7 is a graph showing changes in the degree of vacuum over time when cells according to Example 4 are kept alive in vacuum.

Hereinafter, a mode for carrying out the present invention (hereinafter sometimes referred to as "embodiment") will be described. The present invention is not limited to the following embodiments, and can be implemented with various modifications within the scope of the gist. Furthermore, the embodiments described below are intended to exemplify methods for embodying the technical idea of the present invention, and are not limited to these examples.

The anticancer agent according to the embodiment includes a substance that contains nitrogen 15 and specifically accumulates in cancer cells.

Nitrogen (N) is one of six essential abundant elements (O, C, H, N, Ca, P), accounts for 2.6% of the human body weight, and can be present in all parts of the body. Nitrogen 15 is also written as ¹⁵ N. ¹⁵ N is one of the naturally occurring stable isotopes of nitrogen, and is composed of seven protons and eight neutrons. ^15N accounts for 0.364% of the total nitrogen on Earth. ^15N is also present in the body in the same proportion.

The substance that contains nitrogen-15 and specifically accumulates in cancer cells may be 5-aminolevulinic acid, where the nitrogen is nitrogen-15. 5-Aminolevulinic acid (5-ALA) is synthesized in all cells and is known as a starting material for the porphyrin synthesis pathway. In normal cells, heme, which is necessary for energy metabolism, is synthesized from 5-aminolevulinic acid through a seven-step enzymatic reaction. Heme released from proteins promotes the generation of active oxygen and causes oxidative stress that damages DNA and lipids, so it is rapidly degraded and excreted after energy production. The chemical formula of 5-aminolevulinic acid is as follows.

In the anticancer agent according to the embodiment, N in 5-aminolevulinic acid is substituted with ^15N . 5-Aminolevulinic acid, where the nitrogen is nitrogen 15, is also written as ¹⁵ N_5-ALA. The chemical formula of 5-aminolevulinic acid where nitrogen is nitrogen 15 is as follows.

The anticancer agent according to the embodiment is administered to a human or a non-human animal. Examples of administration routes include, but are not limited to, topical administration, enteral administration including oral administration, and parenteral administration. The administered anticancer drug is taken up by cells.

As shown in FIG. 1, in normal cells, protoporphyrin IX, which is derived from 5-aminolevulinic acid whose nitrogen is nitrogen 15, is converted to heme by iron-adding enzyme (FECH). In normal cells that can normally synthesize heme, nitrogen-15 is excreted and does not accumulate.

However, in cancer cells, induced nitric oxide synthase (iNOS) actively works to maintain a high intracellular nitric oxide (NO) concentration. Therefore, in cancer cells, iron-sulfur complexes rapidly react with nitric oxide (NO) to irreversibly form dinitrosyl dithiolate iron complexes (DNIC), making it impossible to synthesize heme. Therefore, in cancer cells, 5-aminolevulinic acid whose nitrogen is nitrogen 15 and derivatives of 5-aminolevulinic acid whose nitrogen is nitrogen 15, such as protoporphyrin IX whose nitrogen is nitrogen 15, accumulate for a long time.

Therefore, when the anticancer agent according to the embodiment is administered to a human or non-human animal, nitrogen-15 is specifically accumulated in cancer cells, but not in normal cells.

FIG. 2 shows a center-of-mass energy diagram of the proton capture resonance nuclear reaction of the nitrogen-15 nucleus (units are MeV). When a proton and nitrogen 15 collide, when the sum of the binding energy and collision energy of both nuclei matches (resonates with) the excited level of the oxygen 16 nucleus, the proton is captured by the nitrogen 15 nucleus and becomes the oxygen 16 compound nucleus ^16. O ^* is formed. ¹⁶ O ^* in the excited state emits ^{the 4} He ²⁺ nucleus (α ray), which is the most stable among atomic nuclides, and immediately becomes the first excited level of the carbon 12 nucleus, and ^{the 4} . It emits gamma rays of 43 MeV and becomes a ¹² C nucleus in the ground state. This series of resonance nuclear reactions is expressed by the following formula (1).
¹⁵ N( ¹ H, α ₁ γ) ¹² C (1)
Here, 1 of α ₁ represents the first excited level of ¹² C ^* , and the energy difference E ₀ with the excited level of ¹⁶ O ^* is distributed into the kinetic energy of ⁴ He and ¹² C ^* according to the law of conservation of momentum, ⁴ He and ¹² C ^* are released as product ions. If we look at this series of reaction processes in a center-of-gravity system, that is, a coordinate system in which the centers of gravity of protons and nitrogen 15 move at a moving speed V _G , ^{the 16} O ^* complex nucleus is stationary. When the masses of protons and ¹⁵ N and the velocities in the laboratory system are respectively m _H , m _N , u _H , u _N =0, the center of gravity velocity is expressed by the following equation (2).

Figure 3 shows the relationship between the laboratory system and the center of gravity system. The velocities V _H and V _N of the proton and ¹⁵ N in the centroid system are given by u _H -V _G and -V _G , respectively, and the resonance energy ε ₀ is the proton velocity in the laboratory system, as shown in equation (3) below. is equal to the kinetic energy of

The energy difference between the resonance energy level of the ¹⁶ O ^* composite nucleus and the first excited level of ¹² C ^* conserves the momentum of the produced ions, that is, the vector sum of the momentum of each produced ion remains ^{stationary .} The kinetic energy of the generated ions is distributed so that the momentum of the composite nucleus is equal to zero (m _He V _He +m _C V _C =0).

The maximum values of the kinetic energies ε _He and ε _C of ⁴ He and ¹² C ^* in a laboratory system are given by the following equations (5) and (6).

The kinetic energy ε _He , ε _C given by the above equations (5) and (6) is the energy imparted from the ions to the surroundings along the trajectory of the generated ions (linear energy transfer, LET ) and the trajectory of the generated ions. Since LET is proportional to the square of the ion's charge, Z ² , when ⁴ He ²⁺ nuclei with Z=2 and ¹² C ⁶⁺ nuclei with Z=6 are released into the cell as generated ions, ⁴ He ²⁺ Nuclei and ¹² C ⁶⁺ atomic nuclei may have the ability to fly within cells with high kinetic energy and impart high energy to their surroundings, killing cells. FIG. 4 is a graph showing a comparison of the LETs of protons and generated ions ( ⁴ He ²⁺ nucleus and ¹² C ⁶⁺ nucleus). In cancer cells in which the produced ions exhibit a higher LET than protons and have accumulated ¹⁵ N, the cancer cells can be specifically killed by the produced ions due to the ¹⁵ N resonance nuclear reaction.

In addition to the reaction channel of formula (1), there are also reactions that directly de-excite the carbon to the ground state from the excited nuclear level of the ¹⁶ O ^* complex nucleus, and reactions that release only gamma rays without emitting α to generate oxygen 16. There is a reaction that leads to the ground state, and when expressed in formulas, the reaction channels of the following formulas (7) and (8) open simultaneously.
¹⁵ N ( ¹ H, α ₀ ) ¹² C (7)
¹⁵ N ( ¹ H, γ ₀ ) ¹⁶ O (8)

The resonance nuclear reaction represented by formula (8) only emits gamma rays with high penetrating power, and does not cause radiation effects specific to cancer cells, which is the purpose of this embodiment. However, due to the extremely high stability of the ⁴ He ²⁺ nucleus and the ¹² C ^* nucleus, the resonant nuclear reaction channels represented by equations (1) and (7) are more stable than the resonant nuclear reaction channels represented by equation (8). It occurs with an overwhelmingly high probability. The order of nuclear reaction probabilities is expressed by the following equation (9).
(Reaction probability of Equation 1)>(Reaction probability of Equation 7)>>(Reaction probability of Equation 8) (9)

In cancer cells that have accumulated a large amount of nitrogen-15, a resonance nuclear reaction in which protons collide with nitrogen-15 nuclei and generated ions are released occurs with a high probability, making it possible to specifically kill cancer cells.

Figure 5 shows the reaction cross section for each proton resonance energy of ^{the 15} N( ¹ H, α ₁ γ) ¹² C resonance nuclear reaction defined by formula (3), which corresponds to the excited level of the ¹⁶ O ^* complex nucleus. (See Non-Patent Documents 3 and 4.) Figure 6 shows the resonance energy of the proton beam as the residual range from the p-stopping point, which indicates the reaction position of the proton in the body, and the range of the reaction generated ions on the scale of the residual range. . There are 12 resonance energies in the range where a proton enters the body and is attenuated to an energy of 10 MeV or less, a region with a residual range of about 800 μm. At the residual range position corresponding to the resonance energy, the generated ions ⁴ He ²⁺ nucleus and ¹² C ⁶⁺ nucleus are ejected with the kinetic energy defined by equations (5) and (6), and the range of each generated ion is is shown by the radius of a small circle for each resonance energy. High energy is applied to the surrounding area over the range of the generated ion, and the surrounding area is excited with high density. Therefore, cancer cells are damaged at these 12 resonance energy positions, and the cancer cells die. Figure 7 shows the resonance energy of the ¹⁵ N( ¹ H, α ₁ γ) ¹² C resonance nuclear reaction, the residual range of protons in the body, and the reaction product ions ( ⁴ He ²⁺ , ¹² C ⁶⁺ ). The range and biological ionization effect (Bragg's peak) values are summarized below.

In cancer cells given the anticancer agent according to the embodiment, a large amount of protoporphyrin IX, in which nitrogen is nitrogen 15, is synthesized particularly in the mitochondria. Therefore, without being bound by theory, as shown in Figure 8, proton beam irradiation in cancer cells causes many 15N resonance nuclear reactions to occur in the mitochondria, damaging the mitochondria and killing the cancer cells. it is conceivable that.

Therefore, by irradiating a human or non-human animal to which the anti-cancer agent according to the embodiment has been administered with a proton beam, it is possible to specifically kill cancer cells in the body of the human or non-human animal.

There is no particular limitation on the method for producing 5-aminolevulinic acid in which the nitrogen is nitrogen-15. Normal 5-aminolevulinic acid is biosynthesized by feeding the precursors of 5-aminolevulinic acid, glycine and succinic acid, to the fifth mutant strain (CR-520), sixth mutant strain (CR-606), and seventh mutant strain (CR-720), which were created based on the photosynthetic bacterium Rhodobacter sphaeroides IFO12203 and are capable of producing 5-aminolevulinic acid under aerobic dark conditions.

Therefore, by feeding glycine whose nitrogen is 15 and succinic acid whose nitrogen is 15 to a 5-aminolevulinic acid producing bacterium, it is possible to produce 5-aminolevulinic acid whose nitrogen is 15. It is possible.

When producing 5-aminolevulinic acid whose nitrogen is nitrogen 15 during biosynthesis, a large amount of nitrogen is consumed in the synthesis process of proteins, nucleic acids, etc. in addition to the metabolic pathway of 5-ALA, and only 0.364% of nitrogen exists in nature. It is desirable to make effective use of the rare nitrogen 15. To this end, it is desirable to recover a large amount of ¹⁵ N contained in the residue after producing 5-aminolevulinic acid whose nitrogen is 15 through biosynthesis, and return it to the 5-aminolevulinic acid production process. An example of a method for recovering nitrogen from the residue is the Kjeldahl method, in which organic nitrogen is heated in the presence of sulfuric acid and recovered as (NH ₄ ) ⁺ ions. Nitrogen utilization in microalgae, Escherichia coli, and the like can directly take in (NH ₄ ) ⁺ ions, and is suitable as a recovery route for nitrogen-15. The incorporated (NH ₄ ) ⁺ ions are incorporated into the glutamic acid synthesis cycle by glutamine synthetase, and glutamic acid is synthesized. The use of the C5 pathway, in which 5-ALA is synthesized by three types of enzymes (GltX, HemA, and HemL) from glutamic acid synthesized by α-KG (α-ketoglutarate) in the TCA (TriCarboxylic Acid) cycle that takes in glucose, is a biological It can be used in pharmaceuticals as a means to produce 5-aminolevulinic acid where the nitrogen is nitrogen 15.

Substances that contain nitrogen-15 and specifically accumulate in cancer cells are not limited to 5-aminolevulinic acid, where the nitrogen is nitrogen-15. For example, a substance that contains nitrogen-15 and specifically accumulates in cancer cells may be 5-fluorouracil, where the nitrogen is nitrogen-15. Alternatively, the substance that contains nitrogen-15 and specifically accumulates in cancer cells may be a prodrug of 5-fluorouracil, where the nitrogen is nitrogen-15. Examples of prodrugs of 5-fluorouracil where the nitrogen is nitrogen 15 include tegafur where the nitrogen is nitrogen 15, tegafur uracil where the nitrogen is nitrogen 15, tegafur gimeracil oteracil potassium where the nitrogen is nitrogen 15, nitrogen and capecitabine, where nitrogen is nitrogen-15.

Further, the substance containing nitrogen 15 and specifically accumulating in cancer cells according to the embodiment may be a molecular target therapeutic drug whose nitrogen is nitrogen 15. Molecularly targeted therapeutics, for example, have a moiety that binds to cancer cells. Molecularly targeted therapeutic agents, for example, have moieties that bind to biomolecules that are specifically expressed in cancer cells. For example, a portion that binds to a biomolecule specifically expressed in cancer cells may contain nitrogen-15. The moiety that binds to a biomolecule specifically expressed in cancer cells may be an antibody or a portion of an antibody. The molecularly targeted drug may be an antibody-drug conjugate (ADC), where the nitrogen is nitrogen-15, to target and kill tumor cells while sparing healthy cells. The molecular targeting drug may be an antibody or a portion of an antibody whose nitrogen is nitrogen-15, which targets the HER2 (human epidermal growth factor receptor 2) glycoprotein present on the surface of breast cancer cells. An antibody or a portion of an antibody in which the nitrogen is nitrogen-15, which targets HER2, may be obtained, for example, by substituting nitrogen-15 for the nitrogen in trastuzumab (trade name Herceptin).

¹⁵ N used in the cancer treatment drug according to the embodiment is one of six essential elements in large quantities, and can be supplied to all parts of the body.

^{The 15} N used in the cancer treatment drug according to the embodiment only accounts for 0.364% of the total nitrogen in nature, and ^{the 15} N anticancer drug accumulates in cancer cells at a high rate, thereby specifically targeting cancer cells. It is possible to kill it.

¹⁵ N used in the cancer therapeutic drug according to the embodiment is a stable isotope element. If N is replaced with ¹⁵ N in a cancer drug that has already been approved, even if the cancer drug with N replaced with ¹⁵ N is administered to a cancer patient, the burden on the patient will be higher than that of the already approved cancer drug. Equivalent to therapeutic drugs.

The chemical action of ¹⁵ N used in the cancer treatment drug according to the embodiment is completely equivalent to that of nitrogen 14 ( ¹⁴ N), which exists in nature at 99.636%. If an already approved cancer treatment drug contains nitrogen, 99.636% of that nitrogen is ^14N , and even if ^14N is replaced with ^15N at a high concentration, for example at a ratio of 98% or more, The chemical action of the cancer drug in the body is exactly the same, and its toxicity remains the same.

The method of killing cancer cells outside the body according to the embodiment shows the therapeutic effect of a cancer treatment drug containing nitrogen-15. In the method for killing cancer cells outside the body according to the embodiment, in order to make it possible to irradiate cancer cells by specifying the irradiation energy of the proton beam to the resonance energy at which ^{the 15} N resonance nuclear reaction occurs, the cancer cells containing nitrogen 15 are used. It becomes possible to accurately evaluate the therapeutic effects of therapeutic drugs.

The method for killing cancer cells outside the body includes sealing the cancer cells and, optionally, the normal cells together with a culture medium in a vacuum with a polymer film. This makes it possible to keep cells alive in a vacuum chamber through which the proton beam passes.

In this method of killing cancer cells outside the body, the polymer film that seals the cancer cells and, optionally, the normal cells together with the culture medium in a vacuum may be made of polyethylene sulfide material, and is known as Torelina (Toray Industries, Inc.). Commercially available films may also be used. As shown in FIG. 9, polyethylene sulfide film is extremely stable against thermal hydrolysis (see Non-Patent Document 5). Furthermore, as shown in FIG. 10, Torelina has a property of having high permeability to oxygen, nitrogen, and carbon dioxide, but extremely low permeability to water vapor (see Non-Patent Document 5).

Utilizing the extremely stable property of the Torelina film against thermal hydrolysis, it can be used to treat cancer cells and optionally normal cells even in environments where they are exposed to high-density excitation by proton beams while in contact with aqueous culture solutions. This makes it possible to stably seal the entire culture solution in a vacuum. Due to the extremely low water vapor permeability of the Torelina film, water molecules do not turn into water vapor at the interface where the aqueous solution and the Torelina film come into contact, even if the vacuum side surface of the film is under negative pressure due to the extremely high hydrophobicity of the Torelina fibers. The culture solution can exist in a liquid state. While the culture medium maintains a liquid state, highly permeable oxygen and carbon dioxide are maintained dissolved in the liquid, and the oxygen and carbon dioxide necessary for keeping cells alive are in the culture medium. This results in an environment that is maintained in a safe manner.

In order to confine the cancer cells and, optionally, the normal cells together with the culture medium in a vacuum, an instrument having the function of sealing a polymeric film such as Torelina film may be used. The device having the sealing function may be configured to obtain a degree of vacuum necessary for irradiation with a high-energy proton beam, preferably a degree of vacuum of 1×10 ⁻³ Pa or less. The device having the sealing function may be made of soft stainless steel with an extremely low carbon content (0.007% or less), and may have the product name Clean Star B (Daido Steel Co., Ltd.) as a material. The device having the sealing function may have a sealing surface having a semicircular cross section. A highly hard layer of iron nitride is formed on the sealing surface to a thickness of 100 nm on the surface layer by N ₂ ion implantation, and the sealing surface has adhesive properties due to elastic deformation when tightening, and easily peels off when opening. (See Patent Documents 2 and 3).

An anti-cancer agent containing nitrogen 15 is administered to the cancer cells and optionally normal cells prior to placing them into the vacuum vessel. A polymer film such as Torelina film is sealed with the sealing surface, and cancer cells, and optionally normal cells, are kept alive in a vacuum container along with the culture medium. As a result, cancer cells that have taken up ¹⁵ N into their cells together with the anticancer drug, and optionally normal cells, are held in the vacuum container. It is therefore possible to kill cancer cells outside the body by bombarding ¹⁵ N in cancer cells, and optionally normal cells, with protons to create a resonant nuclear reaction.

As shown in FIG. 11, the cancer treatment device according to the embodiment includes an irradiation device 20 that irradiates a human 10 or a non-human animal in which nitrogen 15 has accumulated in cancer cells with a proton beam. The human 10 or non-human animal is administered the above-mentioned anticancer agent or a prodrug of the anticancer agent. The cancer treatment device according to the embodiment may further include an accelerator that accelerates the proton beam. The accelerator may include a laser plasma.

(Example 1)
Rat gastric mucosa-derived cells (RGM-GFP) were prepared as normal cells, and ^rat gastric mucosa-derived cancer cells (RGK-KO) were prepared as cancer cells. The difference was confirmed as follows. Rat gastric mucosa-derived cells and rat gastric mucosa-derived cancer cells are both derived from the stomach and have the same gene sequence, so they are widely used in research on anticancer drugs (see Non-Patent Documents 6 and 7).

RGM-GFP cells and RGK-KO cells were cultured in appropriate culture media for 3 to 4 days to grow to 80% confluence, and then ^15N_5 -ALA was administered, and ^15N per cell was increased according to the elapsed time. The amount of uptake was measured. FIG. 12 shows a schematic diagram of the measurement of ¹⁵ N uptake. ¹⁵ N_5-ALA was administered to RGM-GFP cells and RGK-KO cells that had grown to 80% confluence, and 0 hours, 0.5 hours, 1 hour, 3 hours, 6 hours, 12 hours, and 24 hours after administration. , cell samples were prepared after each period. The cells were washed three times with fresh culture medium to remove ¹⁵ N_5-ALA remaining on the cell surface. Trypsin was added and the cells that had grown attached to the bottom of the Petri dish were peeled off from the Petri dish, and the concentration of released cells was measured.

The concentration was adjusted and 10 ⁵ cells/2 μL were dropped onto a silicon crystal substrate and dried. The sample size was adjusted to approximately 2.5 mm in diameter. The cell sample was set in a sample holder, placed in a vacuum container, and irradiated with a proton beam. Considering that the energy of the proton beam is attenuated inside the sample by the thickness of the sample, ¹⁵ N ( ¹ H, α ₁ γ) ¹² C resonance nuclear reaction (E _r =0.897 MeV) was set at 22 points between 0.894 MeV and 0.994 MeV so that ¹⁵ N inside the sample could be quantified. 4.43 MeV gamma rays emitted by the resonance nuclear reaction with ¹⁵ N were measured with a Bi ₄ Ge ₃ O ₁₂ (BGO) detector.

FIG. 13 shows the measured gamma ray spectrum. Since the main peak at 4.43 MeV and two annihilation gamma ray peaks (3.92 MeVSE.E. and 3.41MeVD.E.) form a broad peak, gamma rays in the energy range of 2.98 MeV to 4.85 MeV The effective gamma ray dose was obtained by integrating the dose and subtracting the natural background (white dots in the figure). Based on the gamma ray dose from a sample into which a ¹⁵ N_5-ALA aqueous solution with a specified concentration was dropped, the amount of ¹⁵ N_5-ALA taken up by cells was determined as an absolute value. The results are shown in FIG. Twenty-four hours after the administration of ¹⁵ N_5-ALA, the amount of ¹⁵ N_5-ALA taken up by cancer cells was more than five times the amount of ¹⁵ N_5-ALA taken up by normal cells.

The above results showed that nitrogen 15 taken into cancer cells as ¹⁵ N_5-ALA is accumulated within the cells and can specifically kill cancer cells.

The specific procedure for quantifying the amount of ¹⁵ N_5-ALA uptake by RGM-GFP cells and RGK-KO cells was as follows.

RGM-GFP and RGK-KO were cultured in a 35 mm dish in an incubator (37° C., 5% CO ₂ ) for 3 to 4 days until 80% confluence.

1000 μL of a medium solution containing 1 mmol/L of ¹⁵ N_5-ALA was added to each dish. Each cell was treated with trypsin at predetermined intervals.

Cells were washed with phosphate-buffered saline (PBS) or 5% glucose and centrifuged three times.

The number of cells was measured using an automatic counting device (Invitrogen Countess (registered trademark)) and adjusted to 1.0×10 ⁵ cells/2 μL. After dropping the cells onto the silicon substrate, after drying, the silicon substrate was set in a vacuum container, and the cells on the silicon substrate were irradiated with a proton beam from a 1 MV tandem accelerator (University of Tsukuba Applied Accelerator Department) to generate resonance nuclei with ¹⁵ N. The 4.43 MeV gamma rays emitted during the reaction were measured with a BGO detector installed outside the vacuum container.

The sapphire plate was irradiated with a proton beam, the beam area was measured from the fluorescence image, and the gamma ray dose normalized to the unit proton beam charge was determined from the relative ratio to the area of the sample dropped onto the silicon substrate.

Intracellular nitrogen 15 was quantified by normalizing the gamma ray dose from a ¹⁵ N_5-ALA aqueous solution sample with a defined concentration to the proton beam charge and comparing the gamma ray dose normalized to the proton beam charge.

The reagents used in Example 1 are as follows.
5-aminolevulinic acid hydrochloride: 018-13133 (Fujifilm Wako Pure Chemical Industries)
¹⁵ N-aminolevulinic acid hydrochloride: N1371 (Medical Isotope)
0.5% Trypsin-EDTA (10x): 15400054
TrypLE Select Enzyme (1X), no phenol red 100ml: 12563011 (Glibco)
10xD-PBS: 048-29805 (Fuji Film Wako Pure Chemical)
D(+)-glucose: 049-31165 (Fujifilm Wako Pure Chemical)

(Example 2)
FIG. 15 shows the genetic structure of pUC4-KIXX (3914 bps), which is a circular artificial plasmid DNA. Since pUC4-KIXX has an ampicillin resistance gene and a kanamycin resistance gene, E. coli transformed with pUC4-KIXX exhibits resistance to ampicillin and kanamycin. Modification in which the N isotope ratio ¹⁵ N/( ¹⁴ N + ¹⁵ N) in pUC4-KIXX was changed stepwise to the natural abundance ratio of 0.364%, 25%, 50%, 75%, and 98% or more. pUC4-KIXX was prepared.

E. coli transformed with each modified pUC4-KIXX was cultured in a medium containing 100 μg/mL ampicillin and a medium containing 20 μg/mL kanamycin, and irradiated with proton beams. As a result, as shown in FIGS ^{. 16 and 17, compared to E. coli transformed with pUC4-KIXX that does not contain 15 N} , E. coli transformed with pUC4-KIXX containing ¹⁵ N is As the isotope ratio increased, the number of bacteria killed by proton beam irradiation increased. Compared to E. coli transformed with pUC4-KIXX that does not contain ¹⁵ N, the number of colonies of E. coli transformed with pUC4-KIXX with a ¹⁵ N isotope ratio of 75% or more is 10 to the third power. decreased by 1 or more.

A large number of E. coli transformed with pUC4-KIXX that does not contain ¹⁵ N was not killed by proton beam irradiation. Therefore, the reason why a large number of E. coli transformed with modified pUC4-KIXX died was that the ampicillin resistance gene and kanamycin resistance gene were damaged by the ¹⁵ N ( ¹ H, α ₁ γ) ¹² C resonance nuclear reaction, and the E. coli drug resistance gene was damaged. This is thought to be due to loss of tolerance.

(Example 3)
Rat gastric mucosa-derived cancer cells (RGK-KO) and normal cells (RGM-GFP) were cultured in 35 mm dishes containing appropriate media for 7 days, and the cells were grown so that all dishes were 80% confluent. I made preparations. ^15N_5 -ALA was added to the medium 24 hours before proton beam irradiation, and the culture conditions in both media were kept the same and maintained for 24 hours to enable comparison with a medium not containing ^15N_5 -ALA. . ¹⁵ RGK-KO cells and RGM-GFP cells cultured in a medium containing N_5-ALA, and RGK-KO cells and RGM-GFP cells cultured in a medium not containing ¹⁵ N_5-ALA were placed in a vacuum together with the culture medium. was held in a sealed state and irradiated with a proton beam having a predetermined ion charge. The sample was irradiated with the proton beam energy at 1.3 MeV in accordance with the 1.21 MeV resonance energy, taking into consideration the energy attenuation of the proton beam due to the culture solution sealed in vacuum and the Torelina film.

FIG. 18 shows a fluorescence micrograph taken immediately (within 3 hours) after proton beam irradiation of RGM-GFP cells cultured in a medium containing no ¹⁵ N_5-ALA. The upper part shows an image of RGM-GFP cells that have been genetically modified to metabolize fluorescent proteins, and the lower part shows photographs of dead cells identified using the DAPI staining method, which stains the nuclear DNA of dead cells. As the irradiation charge amount of the proton beam increases from 2.5nC, 4.0nC, 5.0nC, 10nC, and 25nC, the number of dead cells, that is, the number of cells in the fluorescent protein image (the number of viable cells) and the number of cells in the DAPI-stained fluorescence image, increase. The number of fluorescent protein-imaged cells relative to the sum of the number of cells (number of dead cells) is increasing. RGM-GFP cells cultured in a medium containing no ¹⁵ N_5-ALA were irradiated with a proton beam, and the relationship between the amount of irradiation charge and cell survival rate is shown in FIG. A logarithmic relationship was observed between the two, indicating the characteristics of biological radiation damage caused by proton beams.

FIG. 20 shows photographs immediately after proton beam irradiation of RGK-KO cells cultured in a medium without and a medium containing ^15N_5 -ALA. The photograph in FIG. 20 shows a case where the irradiation charge amount of the proton beam was 4.0 nC for both cells. RGK-KO cells also contain a gene that metabolizes fluorescent proteins, and the cells appear red under a fluorescence microscope. In RGK-KO cells cultured in a medium containing no ¹⁵ N_5-ALA, the number of cells in the fluorescent protein image was 4671, and the number of cells in the DAPI-stained fluorescent image was 3179, and the survival rate was 59.5%. On the other hand, in RGK-KO cells cultured in a medium containing ^15N_5 -ALA, the number of cells in the fluorescent protein image was 4522, the number of cells in the DAPI staining fluorescence image was 1622, and the survival rate was 73.5%. .

FIG. 21 shows a photograph of the cells 24 hours after irradiation with a proton beam at a charge amount of 4.0 nC to RGK-KO cells cultured in a medium containing ¹⁵ N_5-ALA. The survival rates of cells whose survival rates were 73.5% and 85.5% within 3 hours immediately after irradiation decreased to 40.5% and 10.6% after 24 hours, respectively. The tendency for the survival rate of RGK-KO cells to rapidly decrease over time suggests that ^15N_5 -ALA taken up by RGK-KO cells is related to the metabolic pathway induced by protoporphyrin IX in cancer cells. It shows.

(Example 4)
As a method to kill cancer cells outside the body, a vacuum sealing joint (hereinafter referred to as "CS flange") with a sealing function made of Torelina film (4 μm thick, Toray Industries, Inc.) and Clean Star is used. Then, RGM-GFP cells and RGK-KO cells were sealed, set in a vacuum container, and kept in vacuum. In Figure 22, a silicon crystal substrate with a diameter of 15 mm and a thickness of 0.5 mm is placed in a circular dish with a depth of 1.0 mm made of high-purity silicon, and RGM-GFP cells and RGK-KO cells are placed on the substrate together with the culture medium. A photograph of the product being dropped and sealed with Torelina film is shown. A thin carbon film was deposited on one surface of the Torelina film (Toray KP Film Co., Ltd.), making it possible to avoid discharge damage caused by proton beam charging.

When RGM-GFP cells and RGK-KO cells held in vacuum for 20 to 30 minutes were released from the seal and cultured, it was confirmed that both cell types grew again, as shown in the cell photo in Figure 22. , it has been revealed that they can be kept alive under vacuum for a certain period of time.

In Figure 23, RGM-GFP cells and RGK-KO cells are sealed together with the culture solution and evacuated using a dry pump (TSU071E, pumping speed 60 L/s, ultimate vacuum 10 ^-5 Pa, PFEIFFER). It was confirmed that the vacuum dropped at the same speed as the vacuum evacuation process without a cell sample.

Claims (18)

An anticancer agent containing a substance that specifically accumulates nitrogen-15 in cancer cells,
the substance is at least one selected from 5-aminolevulinic acid where the nitrogen is nitrogen 15, and 5-fluorouracil where the nitrogen is nitrogen 15;
An anticancer drug that is administered into a patient's body, and the patient is irradiated with a proton beam after administration. An anticancer agent containing a substance that specifically accumulates nitrogen-15 in cancer cells,
The substance is a molecular target therapeutic drug for cancer cells whose nitrogen is nitrogen-15,
An anticancer drug that is administered into a patient's body, and the patient is irradiated with a proton beam after administration. 3. The anticancer agent according to claim 2, wherein the molecular targeting drug for cancer cells in which the nitrogen is nitrogen-15 includes a moiety that binds to the cancer cells. The anti-cancer agent according to claim 3, wherein the portion that binds to cancer cells is an antibody or a part of an antibody. The anticancer agent according to claim 4, wherein a drug is bound to the antibody or a part of the antibody. The anticancer agent according to claim 4, wherein the antibody or a part of the antibody is trastuzumab in which nitrogen is nitrogen-15. An anticancer drug containing a prodrug of a substance that specifically accumulates nitrogen-15 in cancer cells,
the substance is 5-fluorouracil, where the nitrogen is nitrogen-15;
An anticancer drug that is administered into a patient's body, and the patient is irradiated with a proton beam after administration. 8. The anticancer agent according to claim 7, wherein the prodrug is selected from the group consisting of tegafur, tegafur uracil, tegafur gimeracil oteracil potassium, doxifluridine, and capecitabine, in which nitrogen is nitrogen 15. administering to cancer cells in vitro at least one substance selected from 5-aminolevulinic acid, where the nitrogen is nitrogen 15, and 5-fluorouracil, where the nitrogen is nitrogen 15, to cause the cancer cells to accumulate nitrogen 15;
irradiating the cancer cells with a proton beam outside the body;
A method of killing cancer cells outside the body, including: administering to cancer cells a molecularly targeted therapeutic agent for cancer cells whose nitrogen is nitrogen-15 in vitro, causing the cancer cells to accumulate nitrogen-15;
irradiating the cancer cells with a proton beam outside the body;
A method of killing cancer cells outside the body, including: 11. The method of claim 10, wherein the molecularly targeted therapeutic agent is trastuzumab, where the nitrogen is nitrogen-15. administering a prodrug of 5-fluorouracil in which nitrogen is nitrogen-15 to cancer cells in vitro, causing said cancer cells to accumulate nitrogen-15;
irradiating the cancer cells with a proton beam outside the body;
A method of killing cancer cells outside the body, including: 13. The method of claim 12, wherein the prodrug is selected from the group consisting of tegafur, tegafur uracil, tegafur gimeracil oteracil potassium, doxifluridine, and capecitabine, where the nitrogen is nitrogen 15. Accumulating nitrogen-15 in cancer cells of the non-human animal by administering at least one substance selected from 5-aminolevulinic acid whose nitrogen is nitrogen-15 and 5-fluorouracil whose nitrogen is nitrogen-15 to a non-human animal. to let;
irradiating the non-human animal with a proton beam;
cancer treatment methods, including. administering to a non-human animal a molecular targeting therapeutic agent for cancer cells whose nitrogen is nitrogen-15, thereby accumulating nitrogen-15 in the cancer cells of the non-human animal;
irradiating the non-human animal with a proton beam;
cancer treatment methods, including. 16. The method of claim 15, wherein the molecularly targeted therapeutic agent is trastuzumab, where the nitrogen is nitrogen-15. administering to a non-human animal a prodrug of 5-fluorouracil in which the nitrogen is nitrogen-15, thereby accumulating nitrogen-15 in cancer cells of the non-human animal;
irradiating the non-human animal with a proton beam;
cancer treatment methods, including. 18. The method of claim 17, wherein the prodrug is selected from the group consisting of tegafur, tegafur uracil, tegafur gimeracil oteracil potassium, doxifluridine, and capecitabine, where the nitrogen is nitrogen 15.

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